Compositions and methods for enhancing active agent absorption

ABSTRACT

Compositions and methods for enhancing the absorption of active agents across the mucosa of animal subjects are provided. Methods of administration and appropriate dosage forms are also provided.

The present application is a continuation application of U.S. patentapplication Ser. No. 11/805,202, filed May 21, 2007, which claims thebenefit of U.S. Provisional Patent Application No. 60/747,900, filed onMay 22, 2006, each of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The effectiveness of a drug regimen is often highly dependent onachieving the necessary bioavailability of the drug once it isadministered. Poor bioavailability of a drug makes it necessary toadminister higher doses in order to achieve efficacy, but higher dosagerequirements can lead to toxicity, undesirable side effects, and/ordecreased patient compliance. Although the gastrointestinal tract is acommon route for drug delivery, not all drugs are well absorbed throughthe lining of the gastrointestinal tract. In the case of some drugs,their polar nature or hydrophilic character may make them difficult tobe absorbed. For example, drugs of high molecular weight molecules aredifficult to successfully administer orally, because the acidicconditions and high enzymatic activity in the stomach can degrade suchdrugs. Delivering drugs through other routes, such as buccal,sublingual, rectal, nasal, vaginal mucosa, is one way to providesystemic access for drugs while avoiding the hostile environment of thegastrointestinal tract. However, for this to be successful, the drugadministered must overcome the resistance of these membranes toabsorption. It is therefore clear that any factor that enhances the rateof absorption through such surfaces will result in improved clinicalefficacy of many drug therapies.

Early efforts to enhance the absorption, and therefore bioavailability,of drugs include the use of adjuvants, such as surface active agents,including ionic and non-ionic surfactants to increase biomembranepermeability. However, use of some adjuvants is attended by the risk ofdamaging the biomembrane. Therefore, a need continues to exist forcompositions that safely promote the absorption of poorly absorbeddrugs.

SUMMARY OF THE INVENTION

A composition for enhancing transmucosal absorption of an active agentcan comprise a) at least one active agent; and b) an absorption enhancerhaving the formula:

or a pharmaceutically acceptable salt thereof. In this formula, R₁ canbe null, C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene, arylene, or substitutedalkylene, alkenylene, or arylene. The substituted alkylene, alkenylene,or arylene can be substituted with N, O, S, or P. Further, R₂ can behydrogen, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, aryl, or cycloalkyl; and each R₃can independently be hydrogen, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, or aryl, orsubstituted alkyl, alkenyl, or aryl, said substituted alkyl, alkenyl, oraryl being substituted with N, O, S, or P. X can be a tertiary amine,quaternary amine, tertiary sulfur, carboxylic acid, sulfide, sulfonicacid, sulfenic acid, sulfoxide, phosphoric acid, phosphonic acid,poly(ethylene glycol), saccharide, oligosaccharide, polyol,polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, chitosan, ora combination thereof. A can be null or a coupling moiety such as ether,amide, or ester; B can be null, —CH₂—, O, S, or NH; and Z can be null,O, S, NH, or NR where R is lower alkyl, or wherein Z is joined with R₂to form a heterocyclic ring.

The composition of the present invention may be formulated into variousdosage forms configured to bring the composition into effective contactwith a mucosal surface of a subject, which includes external andinternal mucosal surfaces of the body. Oral or administration can beused to contact the composition with the mucosal surfaces of theintestines, whereas a buccal administration can be used totransmucosally deliver the composition through the mucosal surfaceswithin the oral cavity. Exemplary dosage forms for delivery includetablets, capsules, powders, suspensions, creams, and suppositories.

The present invention is also drawn to methods of enhancing transmucosalabsorption of active agents in a subject, which comprises administeringa composition to the subject so that the composition comes intoeffective contact with a mucosal surface of the subject. The compositioncan be the same as that described above.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

Before particular embodiments of the present invention are disclosed anddescribed, it is to be understood that this invention is not limited tothe particular process and materials disclosed herein as such may varyto some degree. It is also to be understood that the terminology usedherein is used for the purpose of describing particular embodiments onlyand is not intended to be limiting, as the scope of the presentinvention will be defined only by the appended claims and equivalentsthereof.

It is to be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

As used herein, “administration,” and “administering” refer to themanner in which a drug, formulation, or composition is introduced intothe body of a subject. Administration can be accomplished by variousart-known routes such as oral, parenteral, transdermal, inhalation,implantation, etc. Thus, an oral administration can be achieved byswallowing, chewing, or sucking an oral dosage form comprising activeagent(s). Parenteral administration can be achieved by injecting acomposition intravenously, intra-arterially, intramuscularly,intrathecally, or subcutaneously, etc. Transdermal administration can beaccomplished by applying, pasting, rolling, attaching, pouring,pressing, rubbing, etc., of a transdermal preparation onto a skinsurface. Similarly, transmucosal administration can be accomplished byapplying, pasting, rolling, attaching, pouring, pressing, rubbing, etc.,of a transmucosal preparation onto a mucosal surface, as well as byspraying, irrigating the surface with the preparation. These andadditional methods of administration are well known in the art.

The terms “effective amount,” and “sufficient amount” may be usedinterchangeably and refer to an amount of an ingredient which, whenincluded in a composition, is sufficient to achieve an intendedcompositional or physiological effect. Thus, a “therapeuticallyeffective amount” refers to a non-toxic, but sufficient amount of anactive agent, to achieve therapeutic results in treating a condition forwhich the active agent is known to be effective. Various biologicalfactors may affect the ability of a substance to perform its intendedtask. Therefore, an “effective amount” or a “therapeutically effectiveamount” may be dependent on such biological factors. Further, while theachievement of therapeutic effects may be measured by a physician orother qualified medical personnel using evaluations known in the art, itis recognized that individual variation and response to treatments maymake the achievement of therapeutic effects a subjective decision. Insome instances, a “therapeutically effective amount” of an active agentcan achieve a therapeutic effect that is measurable by the subjectreceiving the active agent. The determination of an effective amount iswell within the ordinary skill in the art of pharmaceutical, medicinal,and health sciences.

The term “effective contact” as used herein refer to contact of a natureand duration sufficient to allow a desired effect to be achieved, e.g.effective contact of a preparation with a surface into which it is to beabsorbed will allow a measurable amount, preferably also an effectiveamount, of an agent in the preparation to enter the tissues orbloodstream of a subject, depending on the application target.

As used herein, “carrier” or “inert carrier” refers to typical compoundsor compositions used to carry active ingredients, such as polymericcarriers, liquid carriers, or other carrier vehicles with which abioactive agent, such as insulin, may be combined to achieve a specificdosage form. As a general principle, carriers do not substantially reactwith the bioactive agent in a manner which substantially degrades orotherwise adversely affects the active agent or its therapeuticpotential.

When referring to alkyl, alkylene, alkenyl, alkenylene, or the like, itis understood that these terms include either linear or branchedhydrocarbon chains. These hydrocarbon chains can optionally besubstituted with N, O, S, or P, for example.

The term “null” is used herein to describe variables that, thoughincluded in a compound formulation, can be removed from the formulation.For example, if a variable “B” is said to be null, —CH₂—, O, S, or NH,the variable “B” can be removed from the formulation, or can besubstituted with —CH₂—, O, S, or NH.

According to a more general embodiment of the present invention, acomposition for achieving enhanced absorption of a drug or other activeagent can comprise an effective amount of the active agent itself and anabsorption enhancer or a pharmaceutically acceptable salt thereof. Inone embodiment, the absorption enhancer may have the general formula:X—R₁—Ar—Y_(n)   [Formula I]where Ar can be an aromatic structure such as benzene or naphthalene;Y_(n) can be OH, H, alkyl, aryl, alkenyl, alkynyl, cycloalkyl,carbamidoyl, ester, halogen, halogen-substituted alkyl, amino, alkoxyl,carbamyl, azo, thio, thioalkyl, cyano, sulfone, or alkyl sulfoxidegroups, where n=2-5 of these groups or a combination thereof, and atleast one of which is an OH; R₁ can be null, a saturated or unsaturatedlinear, branched or cyclic alkylene, or arylene, and optionally may besubstituted with functional groups containing hetero-atoms such as N, O,S, or P (R₁ can be coupled to Ar through a coupling moiety); and X caninclude at least one hydrophilic moiety such as an ionizablefunctionality, e.g. tertiary amine, quaternary amine, tertiary sulfur,carboxylic acid, sulfide, sulfonic acid, sulfenic acid, sulfoxide,phosphoric acid, phosphonic acid, or a combination thereof, or otherhydrophilic functionality such as poly(ethylene glycol), saccharides,polyvinyl alcohol, polyacrylic acid, chitosan and derivatives, or acombination of these functionalities.

While not wishing to be bound to a particular theory, it is thought thatthe improvement in absorption that an enhancer provides may be increasedif the enhancer has an appropriate balance of lipophilic and hydrophilicproperties. As can be seen from Formula I, absorption enhancers inaccordance with the present invention can comprise a functional fragmentof a small molecule adjuvant, a lipophilic component, and a hydrophiliccomponent.

In a more specific embodiment, the absorption enhancer can be aderivative of salicylic acid. In one particular embodiment, such anenhancer can have the following formula:

where R₁ can be null, linear, or branched C₁-C₁₂ alkylene, C₂-C₁₂alkenylene, arylene, or substituted alkylene, alkenylene, or arylene,said substituted alkylene, alkenylene, or arylene being substituted withN, O, S, or P; R₂ can be hydrogen or an aliphatic chain such as C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, or an aryl or cycloalkyl functional group; eachR₃ can independently be hydrogen, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, or aryl,or substituted alkyl, alkenyl, or aryl, said substituted alkyl, alkenyl,or aryl being substituted with N, O, S, or P; X can be tertiary amine,quaternary amine, tertiary sulfur, carboxylic acid, sulfide, sulfonicacid, sulfenic acid, sulfoxide, phosphoric acid, phosphonic acid,poly(ethylene glycol), saccharide, oligosaccharide, polyol,polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, chitosan, ora combination thereof. A can be null or a coupling moiety such as amide,ester, or ether; B can be null, —CH₂—, O, S, or NH; and Z can be null,O, S, NH, or NR where R is lower alkyl, or wherein Z is joined with R₂to form a heterocyclic ring. It is noted that the aromatic ring can alsobe naphthalene or another fused aromatic ring structure.

When both B and Z are null, the absorption enhancer may have theformula:

where R₁, R₂, R₃, A, and X represent the same groups as with Formula II.In yet another alternative embodiment, when B is null, the absorptionenhancer has the formula:

where R₁, R₂, R₃, Z, A, and X represent the same groups as with FormulaII.

In a preferred aspect of any of the embodiments represented in FormulasI-IV, R₁ includes an amide, ether, or an ester group by which it isattached to the aromatic ring. In another preferred aspect of theseembodiments, X is COOH.

The absorptive characteristics of the enhancers can also be affected bythe overall lipophilicity of the molecule contributed by R₁ and R₂ aswell as substitution(s) on the aromatic ring. For example, where X—R₁(plus an amide linker moiety or other linker moiety) in Formula II is aHOOCCH₂CH₂CONH (and Z═NH), a more optimal length of R₂ can provide themaximal enhancement in absorbability, while a shorter or longer chainlength may provide a lesser benefit in some circumstances. In addition,absorbability may be affected by the position of the moiety containingR₁ and the amide group of the moiety containing R₂. In the same example,where the moiety containing R₁ is in a para orientation relative to themoiety containing R₂, achieving the maximal absorption enhancement maybenefit from a shorter R₂ chain than when these two moieties are in ameta orientation relative to one another. Accordingly, a preferredlength of an aliphatic chain in R₂ is 3 to 12 carbons, with a still morepreferred length being 4 to 8 carbons when the moiety containing R₁ ispara to moiety containing R₂. When the moiety containing R₁ and moietycontaining R₂ are in meta position, the preferred R₂ chain length may be5 to 10 carbons. In the case when X is a carboxylic acid (HOOC—) andZ—R₂ is NH₂, preferred size of the aliphatic chain length R₁ is 4 to 12carbons when X—R₁ and Z—R₂ are in para position and 5 to12 carbons whenthey are in meta position.

In the salicylate-based enhancers described in Formulas II-IV, the basicstructure of salicylic acid is preserved to the extent that an OH groupis always present at carbon 2 of the ring (and therefore is always orthoto the carbonyl carbon at position 1). Furthermore, the lipophilicfunctionality R₂ extends from the carbonyl, ester, or amide group atposition 1, while R₁ is attached to the ring at any one of carbons 3-6.However, other structural arrangements that will provide both lipophilicand hydrophilic properties are considered within the scope of thisinvention. For example, R₃ may be attached, directly or through anether, ester, or amide linkage, at any of carbons 3-6 instead of R₁,with an alkyl or alkenyl group providing lipophilicity and a hydrophilicgroup (such as carbonyl, nitro, cyano, tri-chloro- ortri-fluoro-substituted groups) providing hydrophilicity. Note, however,that in these arrangements, the relative orientation of the hydroxyl andcarbonyl remains the same (i.e. ortho), as in Formulas II-IV. Also, aswith those previously described embodiments, the length of the carbonchain in R₂ may be adjusted to attain the optimal degree of absorption.

Biologically and chemically active agents that can be made more usefulby enhancing their absorption are exemplified, without limitation, bythe following: growth hormones, including human growth hormones (hGH),bovine growth hormones, and porcine growth hormones; growth hormonereleasing hormones; interferons; cytokines; naturally occurring orrecombinant insulins, including porcine, bovine, and human, optionallyhaving counter ions including sodium, zinc, calcium, and ammonium;insulin-like growth factors, including IGF-1; heparin, includingunfractionated heparin, heparinoids, dermatans, chondroitins, lowmolecular weight heparin, very low molecular weight heparin, andultra-low molecular weight heparin; calcitonin, including salmon, eel,and human; erythropoetin; granulocyte-colony stimulating factor; atrialnaturetic factor; antigens; monoclonal antibodies; somatostatin;protease inhibitors; adrenocorticotropin; gonadotropin releasinghormone; oxytocin; leutinizing hormone releasing hormone; folliclestimulating hormone; glucocerebrosidase; thrombopoietin; humangranulocyte colony stimulating factor; prostaglandins; cyclosporin;vasopressin; cromolyn sodium; vancomycin; desferrioxamine; parathyroidhormone, including its fragments; vitamins; polyethylene glycolderivatives of these compounds, or any combination of these, as well asother proteins, polypeptides, hormones, polysaccharides, carbohydrates,and other organic compounds exhibiting poor bioavailability whenadministered to a subject. Other agents include vaccines, nucleotides,and antiviral agents.

The compositions of the present invention are useful for administeringbiologically or chemically active agents to animals, including but notlimited to birds such as chickens, and mammals such as cows, pigs, dogs,cats, primates, and particularly humans. The present invention may alsobe utilized to deliver agents such as pesticides and hormone disruptorsto insects.

The compositions of the present invention can be particularly useful inenhancing the absorption of active agents across mucosal surfacesoutside of the small and large intestines, so as to provide a viablealternate delivery route to a subject's system other than by oralroutes. Besides avoiding the likely degradation an active agent wouldencounter in the gastrointestinal environment, transmucosaladministration can be a less invasive alternative to oraladministration, due to the relatively easy access to a number of mucosalsurfaces, such as in the buccal cavity, nose, vagina, and rectum. Otherpossible transmucosal routes include the sublingual, ocular,intraduodenal, intracolonic, pulmonary mucosa, jejunum mucosa, and ileummucosa. The compositions of the present invention also encompass bothtraditional oral delivery routes as well as through oral mucosa. Assuch, the present invention also provides methods of enhancingabsorption of active agents in a subject by administering a compositioncomprising an active agent and an enhancer so that it comes intoeffective contact with a mucosal surface of the subject, such as any ofthe surfaces listed above. The dosage form utilized in such a methodwill depend on the mucosal surface involved, and the manner of accessprovided to the surface.

Accordingly, dosage forms for providing enhanced absorption of activeagents by any of these routes can comprise the active agent(s) and anyabsorption enhancer in accordance with the present invention. For oraladministration, dosage forms include liquid suspensions or elixirs,capsules, coated and uncoated tablets, quick dissolving formulations,lozenges, gels, pastes, etc. Buccal administration can include buccalpatches, tablets, lozenges, gels, pastes, etc. Rectal and vaginal dosageforms include liquids, gels, pastes, creams, and suppositories. Nasaladministration may be accomplished with liquids, gels, pastes, or drypowders.

As appropriate to the form utilized, these dosage forms may each alsocomprise excipients in addition to the active agent and enhancer. Forexample, liquid oral dosage forms may include diluents, solubilizers,flavorants, taste masking agents, surfactants, buffering agents,preservatives, antioxidants, colorants, ethanol, propylene glycol,saccharides, and polyethylene glycol, as well as other appropriateadditives known to those skilled in the art, and any combinationthereof. Solid oral dosage forms such as tablets, coated tablets,capsules, and powders may be prepared by conventional skills in the art,and include excipients such as fillers, diluents, disintegrants,lubricants, glidents, plasticizers, colorants, flavorants, taste maskingagents, saccharides, surfactants, binders, buffering agents,preservatives, antioxidants, colorants, polymer coatings, pore formers,and any combination thereof. Formulations for nasal administration mayrequire a propellant. Suppositories may also contain suppository basesto provide proper consistency, such as coconut butter, polyethyleneglycol, etc. Any of these dosage forms may optionally include any of anumber of enzyme inhibitors known in the art such as actinonin orepiactinonin, and derivatives thereof; aprotinin; and Bowman-Birkinhibitor.

EXAMPLES Example 1 Synthesis of methyl 4-aminosalicylate

4-Aminosalicylic acid (12.3 g) is placed in a round bottom flask. Theflask is charged with 150 mL of methanol and 20 mL of concentratedsulfuric acid. The reaction mixture is brought to gentle reflux for 1day, and then allowed to cool to room temperature. The mixture isfurther chilled in a freezer and methyl 4-aminosalicylate bisulfate iscrystallized. The crystals are collected and washed with cold methanolto afford 15.8 g of the product as off-white needles.

Example 2 Synthesis of methyl4-(3-carboxy-propionylamino)-2-hydroxybenzoate

Methyl 4-aminosalicylate bisulfate (5 g) is placed in a 50-mL roundbottom flask. The flask is charged with 20 mL of methylene chloride and15 mL of triethylamine. Finally, succinic anhydride (2.75 g) is added.The reaction mixture is then allowed to stir at ambient conditionsovernight. Solvents are removed under reduced pressure, yielding aviscous residue, to which 1 N HCl is added.Methyl(3-carboxy-propionylamino)-2-hydroxybenzoate is yielded as tanpowder. The powders are collected by filtration, washed with water, anddried.

Example 3 Synthesis of N-n-hexyl4-(3-carboxy-propionylamino)-2-hydroxybenzamide

Methyl 4-(3-carboxy-propionylamino)-2-hydroxybenzoate from Example 2 (1g) is dissolved in 15 mL of methanol. n-Hexylamine (5 mL) is added tothis solution, and the reaction mixture is brought to mild reflux for 2days. Solvents are removed under vacuum and residue acidified using 1 NHCl. N-n-hexyl 4-(3-carboxy-propionylamino)-2-hydroxybenzamideprecipitate is collected, washed with water, and dried.

Example 4 Synthesis of 4-(3-carboxy-propionylamino)-2-hydroxybenzamide

Methyl 4-(3-carboxy-propionylamino)-2-hydroxybenzoate (1 g) from Example2 is dissolved in 5 mL of 28% ammonium hydroxide solution. The reactionmixture is stirred at ambient conditions for 16 hours. In an ice-waterbath, the solution is carefully acidified using concentratedhydrochloric acid. The precipitate is collected by filtration, washedwith water and dried.

Example 5 Synthesis of 4-aminosalicylamide

Methyl 4-aminosalicylate bisulfate (5 g) is suspended in 20 mL ofmethanol. To the solution, 20 mL of 28% ammonium hydroxide is addedslowly until all solid is dissolved. The reaction mixture is stirred atambient conditions overnight. Solvent is removed under reduced pressureand water is lyophilized. The residue is collected, washed with minimumamount of ice water and dried to yield 2.5 g of the product as off-whitepowders.

Example 6 Synthesis of 4-(9-carboxy-nonanoylamino)-2-hydroxybenzamide

4-aminosalicylamide (2 g) from Example 5 is charged with 20 mL ofmethylene chloride. To the solution, 3.3 g of methyl10-chloro-10-oxodecanoate is added. The reaction is initiated byaddition of 2.6 mL of triethylamine. The reaction mixture is allowed tostir at ambient conditions for 2 days. Solvent is removed and theresidue is acidified using 1 N HCl. The precipitate is extracted intoethyl acetate. The organic layer is further extracted with 1 N NaOH andsaturated NaCl solutions. After drying over anhydrous sodium sulfate,solvent is removed. The residue is reconstituted in tetrahydrofuran (10mL) and 10 mL of 2 N NaOH is added. The solution is brought to gentlereflux for 2 hours. The disappearance of the methyl ester is followed byHPLC. When all ester is hydrolyzed, the solution is cooled and acidifiedand the product is extracted into ethyl acetate. The organic layer iswashed with saturated NaCl and dried over anhydrous sodium sulfate.Solvent is removed under reduced pressure and the product is obtained asoff-white powder.

Example 7 Synthesis of methyl 2,5-dihydroxybenzoate

2,5-Dihydroxybenzoic acid (4 g) is dissolved in 20 mL of methanol. Tothe solution, 3 mL of concentrated sulfuric acid is added. The reactionmixture is brought to gentle reflux over 24 hours. Methanol is removedand the residue is diluted with water. The compound of interest isextracted into ethyl acetate. The organic layer is extracted withsaturated sodium bicarbonate solution and dried over anhydrous sodiumsulfate. Solvent is removed and the product is in the form of off-whitepowders.

Example 8 Synthesis of N-n-hexyl 2,5-dihydroxybenzamide

Methyl 2,5-dihydroxybenzoate (1 g) is dissolved in 10 mL ofn-hexylamine. After overnight heating in an oil bath (60° C.), themixture is cooled to room temperature. n-Hexylamine is removed underreduced pressure. The residue is dissolved in ethyl acetate and furtherextracted with 1 N HCl. The organic layer is extracted with saturatedsodium bicarbonate and dried over anhydrous sodium sulfate. Solvent isremoved under reduced pressure to yield the product as an off-whitepowder.

Example 9 Synthesis of N-n-hexyl5-(3-carboxy-propionyloxy)-2-hydroxybenzamide

N-n-Hexyl 2,5-dihydroxybenzamide (1 g) is dissolved in 15 mL ofmethylene chloride. To the solution 0.75 g of succinic anhydride and0.95 g of 4-N,N-dimethylaminopyridene is added. The reaction mixture isallowed to stir at ambient conditions over 24 hours. Solvent is removedand the residue is reconstituted in ethyl acetate and extracted twicewith 1 N HCl to remove the catalyst. The organic layer is furtherextracted with saturated sodium chloride solution and dried overanhydrous sodium sulfate. Solvent is removed under reduced pressure toyield the product as an off-white powder.

Example 10 Synthesis of n-hexyl 2,4-dihydroxybenzoate

2,4-Dihydroxybenzoic acid (5 g) is added to 20 mL of n-hexanol.Concentrated sulfuric acid (5 mL) is added dropwise. The reactionmixture is heated at 60° C. for 6 days. Hexanol is removed under reducedpressure. The residue is recrystalized from methanol/water mixture. Thecrystals are collected, washed with cold methanol and dried.

Example 11 Synthesis of n-hexyl4-(3-carboxy-propionyloxy)-2-hydroxybenzoate

n-Hexyl 2,4-dihydroxybenzoate (2 g) is dissolved in 25 mL of methylenechloride. To the solution, 0.85 g of succinic anhydride and 1.05 g of4-N,N-dimethylpyridene are added. The reaction mixture is allowed tostir at ambient conditions overnight. The reaction mixture is extractedwith 1 N HCl, water and saturated sodium chloride solution. The organiclayer is dried over anhydrous sodium sulfate. Solvent is removed underreduced pressure to yield the product as an off-white powder.

Example 12 Enhancement of Bioavailability of Insulin Using AbsorptionEnhancer

In a vial, 0.5 g of N-n-hexyl4-(3-carboxy-propionylamino)-2-hydroxybenzamide from Example 3 issuspended in 2 mL of water. It is neutralized by 1 equivalent of sodiumhydroxide. The pH is adjusted to 7-8 by 1N HCl and saturated sodiumbicarbonate. Bovine pancreatic insulin (1.75 mL; Sigma, 10 mg/mL; in pH8.2 HEPES buffer) is added and mixed by gentle swirling. The solutiondensity is determined so that the required dose may be calculated.

The solution is then given to Sprague-Dawley rats (2-day fasting,female, 250-300 g, n=3), intracolonically. Blood samples are taken fromthe tail vein and glucose levels measured using an OneTouch® UltraSmart®blood glucose meter. The analysis yields a blood glucose profile inwhich increasing the dose of absorption enhancer results in greaterdecreases in blood glucose, as shown in Table 1.

TABLE 1 Blood glucose depression (in % of basal level) induced byintracolonically administrated bovine pancreatic insulin using N-n-hexyl4-(3-carboxy-propionylamino)-2-hydroxybenzamide as the absorptionenhancer Dose of enhancer Time % Glucose Depression From Basal Level(min) 0 mg/kg 5 mg/kg 25 mg/kg 100 mg/kg 200 mg/kg 0 100 100 100 100 10030  94 ± 4 78 ± 9 52 ± 7 50 ± 8 46 ± 4 60 102 ± 7 83 ± 3 28 ± 3 29 ± 328 ± 3 90 105 ± 3 77 ± 5 29 ± 3 20 ± 2 33 ± 3 120 107 ± 7 99 ± 4 31 ± 819 ± 3 25 ± 3 150  94 ± 7 105 ± 6  29 ± 6 29 ± 6 26 ± 4 180 111 ± 7 96 ±7 47 ± 8 22 ± 2 33 ± 2 210 113 ± 4 113 ± 8 67 ± 5 30 ± 6 29 ± 4 240 100± 7 105 ± 6 77 ± 5 49 ± 5 54 ± 7

Examples 13-22

The general procedure followed in Example 12 above is followed inExamples 13-22 (using different compounds and different dosages as setforth below), with the following results.

TABLE 2 Blood glucose depression from basal level as a function ofabsorption enhancer structure and dose Enhancer* 50% Example X—R₁ (withamide Dose Insulin depression No. Formula coupling moiety) Z—R₂ mg/Kgmg/Kg at 60 min. 13 IV 4-HOOC(CH₂)₂CONH NH₂ 200 7 negative 14 IV4-HOOC(CH₂)₂CONH NHC₂H₅ 200 7 negative 15 IV 4-HOOC(CH₂)₂CONH NHC₄H₉ 1007 positive 16 IV 4-HOOC(CH₂)₂CONH NHC₆H₁₃ 25 7 positive 17 IV4-HOOC(CH₂)₂CONH NHC₈H₁₇ 25 7 positive 18 IV 4-HOOC(CH₂)₂CONH NHC₁₀H₂₁50 7 positive 19 IV 4-HOOCCH₂CH(CH₃)CH₂CONH NHC₆H₁₃ 200 7 positive 20 IV4-HOOC(CH₂)₂CONH OCH₃ 200 7 negative 21 IV 4-HOOC(CH₂)₂CONH OC₂H₅ 200 7negative 22 IV 4-HOOC(CH₂)₂CONH OC₄H₉ 50 7 positive *R₃ for each of theabove enhancers is “H” (hydrogen), accordingly the variable is not shownin the table.

It is noted that though Examples 13, 14, 20, and 21 are shown as“negative,” that does not mean that no enhancement occurred. It onlymeans that a 50% reduction in glucose levels was not reached in 60minutes. As insulin reduces glucose levels in the blood, a depression inglucose levels indicates insulin reaching the blood efficaciously.

While the forgoing examples are illustrative of the principles of thepresent invention in one or more particular applications, it will beapparent to those of ordinary skill in the art that numerousmodifications in form, usage and details of implementation can be madewithout the exercise of inventive faculty, and without departing fromthe principles and concepts of the invention. Accordingly, it is notintended that the invention be limited, except as by the claims setforth below.

What is claimed is:
 1. A composition, comprising: a) at least one activeagent selected from the group consisting of a polypeptide, a hormone, apolysaccharide, prostaglandins, cyclosporin, cromolyn sodium,vancomycin, desferrioxamine and vitamins; and b) an absorption enhancerhaving the formula:

or a pharmaceutically acceptable salt thereof, wherein R₁ is null,C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene, arylene, or substituted alkylene,alkenylene, or arylene, said substituted alkylene, alkenylene, orarylene being substituted with N, O, S, or P; R₂ is C₁-C₁₂ alkyl, C₂-C₁₂alkenyl, aryl, or cycloalkyl; each R₃ is independently hydrogen, C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, or aryl, or substituted alkyl, alkenyl, or aryl,said substituted alkyl, alkenyl, or aryl being substituted with N, O, S,or P; wherein the composition is in a dosage form configured to bring atherapeutically effective amount of the at least one active agent intoeffective contact with a mucosal surface of a subject.
 2. Thecomposition of claim 1, wherein the at least one active agent isselected from the group consisting of growth hormones, growth hormonereleasing hormones, interferons, cytokines, insulins, insulin-likegrowth factors, heparins, calcitonins, erythropoetin, granulocyte-colonystimulating factor, atrial naturetic factor, antigens, monoclonalantibodies, somatostatin; protease inhibitors, adrenocorticotropin,gonadotropin releasing hormone, oxytocin, leutinizing hormone releasinghormone, follicle stimulating hormone, glucocerebrosidase,thrombopoietin, human granulocyte colony stimulating factor,valinomycin, cyclosporin, vasopressin, cromolyn sodium, vancomycin,parathyroid hormone, polyethylene glycol derivatives of these compounds,and combination thereof.
 3. The composition of claim 1, wherein themoiety containing R₁ is para on the aromatic ring with respect to themoiety containing R₂.
 4. The composition of claim 1, wherein the moietycontaining R₁ is meta on the aromatic ring with respect to the moietycontaining R₂.
 5. The composition of claim 1, further comprising anenzyme inhibitor.
 6. The composition of claim 1, wherein the activeagent is selected from the group consisting of proteins, polypeptides,hormones, polysaccharides, carbohydrates, and combinations thereof. 7.The composition of claim 1, formulated into a dosage form selected fromthe group consisting of tablet, capsule, lozenge, liquid solution,liquid suspension, paste, gel, cream, powder, patch, and suppository. 8.The composition of claim 1, wherein the dosage form is a liquid solutionor suspension.
 9. The composition of claim 1, wherein the dosage form isa patch.
 10. The composition of claim 1, wherein the dosage form is asuppository.
 11. The composition of claim 1, formulated in an oraldosage form.
 12. The composition of claim 1, wherein the dosage formcomprises an additive selected from the group consisting of diluents,solubilizers, excipients, flavorants, taste masking agents, surfactants,buffering agents, preservatives, antioxidants, colorants, fillers,disintegrants, lubricants, glidants, plasticizers, ethanol, poreformers, propylene glycol, saccharides, polyethylene glycol, andcombinations thereof.
 13. A method of enhancing transmucosal absorptionof active agents in a subject, comprising administering a composition tothe subject so that the composition comes into effective contact with amucosal surface of the subject, said composition comprising: a) at leastone active agent selected from the group consisting of a polypeptide, ahormone, a polysaccharide, prostaglandins, cyclosporin, cromolyn sodium,vancomycin, desferrioxamine and vitamins; and b) an absorption enhancerhaving the formula:

or a pharmaceutically acceptable salt thereof, wherein R₁ is null,C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene, arylene, or substituted alkylene,alkenylene, or arylene, said substituted alkylene, alkenylene, orarylene being substituted with N, O, S, or P; R₂ is C₁-C₁₂ alkyl, C₂-C₁₂alkenyl, aryl, or cycloalkyl; each R₃ is independently hydrogen, C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, or aryl, or substituted alkyl, alkenyl, or aryl,said substituted alkyl, alkenyl, or aryl being substituted with N, O, S,or P.
 14. The method of claim 13, wherein the mucosal surface isselected from the group consisting of buccal, sublingual, nasal, rectal,pulmonary, ocular, or vaginal.
 15. The method of claim 13, wherein thecomposition is administered orally.
 16. The method of claim 13, whereinthe composition is administered in a dosage form selected from the groupconsisting of tablet, capsule, lozenge, liquid solution, liquidsuspension, paste, gel, cream, powder, patch, and suppository.
 17. Themethod of claim 13, wherein the dosage form is a liquid solution orsuspension.
 18. The method of claim 13, wherein the dosage form is atablet or capsule.
 19. The method of claim 13, wherein the dosage formis a suppository.
 20. The method of claim 13, wherein the dosage form isa cream.
 21. The method of claim 13, wherein the active agent isselected from the group consisting of growth hormones, growth hormonereleasing hormones, interferons, cytokines, insulins, insulin-likegrowth factors, heparins, calcitonins, erythropoetin, granulocyte-colonystimulating factor, atrial naturetic factor, antigens, monoclonalantibodies, somatostatin; protease inhibitors, adrenocorticotropin,gonadotropin releasing hormone, oxytocin, leutinizing hormone releasinghormone, follicle stimulating hormone, glucocerebrosidase,thrombopoietin, human granulocyte colony stimulating factor,valinomycin, cyclosporin, vasopressin, cromolyn sodium, vancomycin,parathyroid hormone, polyethylene glycol derivatives of these compounds,and combination thereof.